EZH2 Mediates Epigenetic Silencing of Neuroblastoma Suppressor Genes CASZ1, CLU, RUNX3 and NGFR

Author Manuscript Published OnlineFirst on November 8, 2011; DOI: 10.1158/0008-5472.CAN-11-0961 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

EZH2 mediates epigenetic silencing of neuroblastoma

suppressor genes CASZ1, CLU, RUNX3 and NGFR

Chunxi Wang1, Zhihui Liu1, Chan-Wook Woo1, Zhijie Li 1, Lifeng Wang5, Jun S. Wei2,

Victor E. Marquez3, Susan E. Bates4, Qihuang Jin5, Javed Khan2, Kai Ge5 and Carol J.

Thiele1*

1Cell & Molecular Biology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD 2Oncogenomics Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD 3Chemical Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD 4 Molecular Therapeutics Section, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD 5Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney, National Institutes of Health, Bethesda, MD

*: Address correspondence to: Carol J. Thiele (E-mail: [email protected], Tel: 301-496-1543)

Running title: Tumor suppressor genes are regulated by EZH2 in NB

Keywords: CASZ1, neuroblastoma, EZH2, NGFR, CLU, RUNX3

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 8, 2011; DOI: 10.1158/0008-5472.CAN-11-0961 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with an

undifferentiated status and generally poor prognosis, but the basis for these characteristics

remains unknown. In this study, we show that upregulation of the Polycomb complex

histone methytransferase EZH2, which limits differentiation in many tissues, is critical to

maintain the undifferentiated state and poor prognostic status of NB by epigenetic

repression of multiple tumor suppressor genes. We identified this role for EZH2 by

examining the regulation of CASZ1, a recently identified NB tumor suppressor gene

whose ectopic restoration inhibits NB cell growth and induces differentiation. Reducing

EZH2 expression by RNAi-mediated knockdown or pharmacological inhibiton with 3-

deazaneplanocin A (DZNep) increased CASZ1 expression, inhibited NB cell growth and

induced neurite extension. Similarly, EZH2-/- mouse embryonic fibroblasts (MEFs)

displayed 3-fold higher levels of CASZ1 mRNA compared to EZH2+/+

MEFs. In cells with increased expression of CASZ1, treatment with HDAC inhibitors

decreased expression of EZH2 and the Polycomb complex component SUZ12. Under

steady-state conditions H3K27me3 and PRC2 components bound to the CASZ1 gene

were enriched, but this enrichment was decreased after HDAC inhibitor treatment. We

determined that the tumor suppressors CLU, NGFR and RUNX3 were also directly

repressed by EZH2 like CASZ1 in NB cells. Together, our findings establish that

aberrant upregulation of EZH2 in NB cells silences several tumor suppressors, which

contribute to the genesis and maintenance of the undifferentiated phenotype of NB

tumors.

Precis

Dysregulation of a single targetable histone methyltransferase is found to be a core

contributor to neuroblastoma phenotypes, highlighting a novel general approach to treat

this common and deadly pediatric tumor.

Introduction

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood,

and accounts for 15% of all pediatric oncology deaths (1-3). Biological and genetic

features have been used to stratify patients for risk and more intensive treatment.

Biological features such as tumor histopathology or the Shimada classification system

have revealed that undifferentiated or stroma-poor tumors have a worse prognosis than

ganglioneuromas, which are mature, stroma rich tumors containing more differentiated

ganglion cells. Patients with ganglioneuroblastomas, tumors that contain a spectrum of

elements seen in both undifferentiated as well as well-differentiated tumors, have variable

prognoses. Recent microarray analyses indicate the transcriptome of tumors from patients

with poor prognoses is enriched in cell cycle related genes, while that of tumors from

patients who have favorable prognoses tumors is enriched in differentiation-associated

genes (4). The mechanisms by which the genetic alterations associated with

neuroblastoma contribute to the undifferentiated state of NB are still unknown.

A number of genetic alterations are associated with NB and are thought to affect

the differentiation potential of the pluripotent sympathetic neuroblasts from which

neuroblastomas are thought to arise. Patients whose neuroblastoma tumors contain

genomic MYCN amplification and allelic deletion of the short arm of chromosome 1

Tumor suppressor genes are regulated by EZH2 in NB 3

(Chr1p deletion) have the highest risk and worst prognoses (reviewed in (2)). Chr1p

deletion is highly correlated with MYCN amplification (5). Loss of heterozygosity of

1p36 (1p36LOH) is found in 20%-40% cases and is independently associated with

progression-free survival (6). The consistent loss of 1p36 brought attention to this region

as the site of a putative NB tumor suppressor gene (2, 3). Genes in the 1p36 region such

as CHD5 (7, 8), miR-34a (9), TP73 (10) and most recently CASZ1, a neuronal

differentiation gene, have been shown to possess tumor suppressor activity (11).

CASZ1 is the human homologue of the drosophila zinc finger transcriptional

factor Castor. Drosophila castor (cas) is specifically expressed in a subset of central

nervous system (CNS) neuroblasts, and regulates late stage neurogenesis and neural fate

determination (12). Liu et.al report that the level of human CASZ1 expression increases

upon the induction of differentiation in NB cells and mesenchymal cells (13). The

restoration of CASZ1 expression in NB cells inhibits cell proliferation in vitro and

decreases tumor growth in vivo (11). In an evaluation of primary NB tumors, the

expression of CASZ1 is significantly decreased in aggressive NB compared with the

favorable tumors (14, 15). The finding that no tumor-associated nucleotide mutation is

found in the coding sequence of CASZ1 (15, 16) suggests that mechanisms such as

epigenetic silencing may be involved in the decreased expression of CASZ1 in tumors of

patients with unfavorable prognoses.

Major mechanisms of epigenetic silencing of gene expression include regulation

of DNA methylation and the posttranslational modifications of histones. DNA

methylation on the 1p36 region has been shown to mediate silencing of CHD5 in NB

tumors cells (8). However, no consistent CpG methylation site in the 5’ proximal region

Tumor suppressor genes are regulated by EZH2 in NB 4

or first intron of CASZ1 has been identified in either NB cell lines or primary tumors that

differs from normal tissues (11, 15, 16). Thus it is unlikely that DNA methylation

accounts for low CASZ1 expression in NB cells. The findings that the histone

deacetylase inhibitors, depsipeptide (11) and trichostatin A (15) induce CASZ1

expression in NB cells, suggest that suppressive histone modifications inhibit CASZ1

gene expression.

Histone acetylation tightly associates with gene activation and the trimethylation

of histone 3 on lysine 27 (H3K27me3) is a well-known histone mark associated with

gene silencing. H3K27me3 is mediated by the methyltransferase EZH2, which is the

enzymatically active component of the Polycomb Repressor Complex 2 (PRC2) (17).

PRC2 contains three core subunits, enhancer of zeste 2 (EZH2), embryonic ectoderm

development (EED) and suppressor of zeste 12 homolog (SUZ12) (reviewed in (18-20)).

EZH2 is essential for stem cell identity and pluripotency (reviewed in (18-20)). PRC2

regulates a large set of developmental genes in embryonic stem cells, such as the HOX

gene clusters, SOX, PAX and WNT gene families. In retinoic acid (RA) induced neural

stem cell differentiation, EZH2 expression is decreased in differentiated neural cells,

consistent with decreased binding of EZH2 to RA-inducible target genes (reviewed in

(18). While PRC2 is released from genes (HOXA 1-5, ZIC1, CKM) expressed during the

differentiation, it is also recruited to the certain genes (HOXA9-13, Neroug2, Olig2) that

may be suppressed in specific cell lineages (reviewed in (19)). This dynamic recruitment

and displacement of PRC2 together with the tissue specific transcriptional factors

determines cell lineage (reviewed in (19)).

Tumor suppressor genes are regulated by EZH2 in NB 5

Over-expression of EZH2 is found in a number of different cancers and is

associated with the progression of prostate (21, 22), breast (23), Ewing’s sarcoma (24)

and glioblastoma (25). The oncogenic function of EZH2 is partially attributable to the

ability of the PRC2 to localize to a number of well-known tumor suppressor genes, such

as INK4A/B (26, 27), E-cadherin (28) and RUNX3 (29).

Until now, the function of the PRC2 and EZH2 has not been evaluated in NB. In

this study, we identify that NB patients with a poor prognoses have increased levels of

EZH2 mRNA. Moreover we find that silencing of EZH2 leads to decreased H3K27me3

and increased expression of the NB tumor suppressor CASZ1, which is consistent with a

model in which one allele of the CASZ1 may be lost by 1p LOH while remaining

allele(s) are subject to epigenetic silencing by EZH2 mediated H3K27me3. Furthermore,

we find that EZH2 silences a number of tumor suppressors, which control differentiation

in NB such as CLU, RUNX3 and NGFR in NB cells. Finally we find that the genetic or

pharmacologic inhibition of EZH2 inhibits NB cell growth and induces differentiation.

Tumor suppressor genes are regulated by EZH2 in NB 6

Material and Methods

Cell culture, transduction and Reagents

Neuroblastoma cell lines used in this study are listed in Supplemental Table 1. NB

cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2mM

glutamine, and 100g/ml of penicillin/streptomycin at 37°C in 5% CO2. The

immortalized EZH2 -/- MEF cells and control MEF cells were constructed as described

(30), and cultured in DMEM containing 10% fetal bovine serum, 2mM glutamine, and

100g/ml of penicillin/streptomycin at 37°C in 5% CO2. For experiments, depsipeptide,

3-deazaneplanocin A (DZNep), and Z-VAD-FMK (R&D Systems Inc, Minneapolis, MN)

were diluted to the indicated concentrations in cell culture media. SMS-KCNR cells were

plated onto 100-mm dishes at a density of 5 million cells per dish. After 24 hrs, cells

were infected by EZH2 or non-target shRNA lentiviral particles (Sigma-Aldrich Corp,

Saint Louis, MO) following the manufacturer’s protocol. The cell viability was checked

using the Cell-Titer Blue assay Kit or MTS assay (Promega, Madison, WI). Caspase 3/7

activities were evaluated using the Caspase-Glo 3/7 assay kit (Promega, Madison, WI).

RNA preparation and quantitative Real-time PCR

Cells were detached mechanically, washed with ice-cold PBS, and processed for RNA

extraction using the RNeasy kit (Qiagen Inc., Chatsworth, CA). qRT-PCR was performed

on ABI Prism 7000 (Applied Biosystems, Carlsbad, CA) to check the EZH2, β2MG,

RUNX3, CLU, NGFR and CASZ1 expression using Maxima® Probe/ROX qPCR Master

Mix or Maxima® SYBR Green/ROX qPCR Master Mix) (Fermentas Inc, Glen Burnie,

Tumor suppressor genes are regulated by EZH2 in NB 7

MD). The samples were normalized to the levels of β-microglobulin (β2MG). The primer

sets are listed in the Supplemental Table 2.

Protein assays

Cells were cultured and harvested as above, and nuclear protein extractions were

prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher

Scientific Inc., Rockford, IL) according to the manufacturer’s protocol. Immunologic

detection of protein expression utilized the following antibodies: anti-EZH2 (BD,

Franklin Lakes, NJ), anti-SUZ12, anti-EED (Abcam Inc, Cambridge, MA), anti-KDM6A

(31), anti-H3K27me3, ant-H3K27Ac (Millipore Corp., Billerica, MA), and anti-H3 C-

terminal (Active motif, Carlsbad, CA). Immunoreactivity was determined using the

enhanced chemiluminescence method (Thermo Fisher Scientific Inc., Rockford, IL).

Chromatin immunoprecipitation assay

SMS-KCNR, SH-SY5Y and NGP cells were plated into 15cm dishes at a density

of 5×106 cells per dish and treated with depsipeptide (2ng/ml). After 24 hours treatment,

ChIP assays were carried out as described (30). For each ChIP assay, the following

antibodies (2ug) were used; EZH2 (Active motif, Carlsbad, CA), SUZ12, EED (Abcam

Inc, Cambridge, MA), H3K27me3, H3K27ac, RNA polymerase II (Millipore Corp.,

Billerica, MA) or IgG control (Santa Cruz Biotechnology, Santa Cruz, CA). ChIP

enriched DNA and input DNA were subjected to qRT-PCR analysis with Maxima®

SYBR Green/ROX qPCR Master Mix (Fermentas Inc, Glen Burnie, MD). Enrichment by

ChIP assay on the specific genomic regions was assessed relative to the input DNA. The

primer sets are listed in the Supplemental Table 3.

Xenograft model

Tumor suppressor genes are regulated by EZH2 in NB 8

SMS-KCNR cells were resuspended in Hanks balanced salt solution and Matrigel

(Trevigen, Gaitherburg, MD) and 100ul cell suspension containing 2×106 cells was

placed in the subcutaneous tissue of the right flanks of 4-5 week old nude mice (Taconic,

Germantown, NY). Treatment was initiated when tumors reached approximately

100mm3. Cohorts of mice (7 mice/group) received DZNep (2.5mg/kg) or vehicle control

(normal saline) twice daily, 3 days per week. The dimensions (length (L) and width (W))

of the tumors were assessed three times a week and the volume was calculated as

(L×W2)/2. Student’s t test was used to compare the tumor volume between the DZNep

and non-treatment groups and p values less than 0.05 were considered significant.

Results

EZH2 expression is associated with NB outcome

Since EZH2 is deregulated in a wide variety of cancers (21-23), we first analyzed

the expression of EZH2 in NB patient tumor samples. According to the histopathological

features, NB can be divided into Neuroblastoma (undifferentiated, schwannian stroma-

poor, high risk), Gangioneuroblastoma and Ganglioneuroma (differentiated ganglion

cells, schwannian stroma-rich, low-risk). Using the Oncomine microarray database

(www.oncomine.com), we found that the expression level of EZH2 was higher in

Neuroblastoma compared to Ganglioneuroblastoma and Ganglioneuroma (Fig 1A, left

panel) (32). Consistently EZH2 is expressed at a higher level in stroma-poor tumors

compared to stroma-rich tumors (Fig 1A, right panel). Patients, whose neuroblastoma

tumors are stroma-poor or have an undifferentiated histopathology, have a worse

prognosis (reviewed in (1, 3)). This suggests that EZH2 expression may be associated

Tumor suppressor genes are regulated by EZH2 in NB 9

with NB prognosis. The Kaplan-Meier plots of overall survival, downloaded from the R2

microarray analysis and visualization platform (http://hgserver1.amc.nl/cgi-

bin/r2/main.cgi?&species=hs), demonstrated that high expression of EZH2 was

associated with poor prognosis (p=0.00042 where the cut-off value was chosen by

smallest p value), while high expression of CASZ1 was associated with good prognosis

(p=0.0011, where the cut-off value was chosen by smallest p value) (Fig 1B). Using

another dataset (33) containing 168 clinical NB tumor samples and 13 NB cell lines we

extracted the median-centered log-2 values for expression of CASZ1 and EZH2 which

were clustered and color-coded using CIMminer software (Fig 1C). The samples with

high expression of EZH2 have low expression of CASZ1, while low expression of EZH2

was more frequently associated with high expression of CASZ1. The correlation between

EZH2 and CASZ1 expression was statistically significant (ρ=-0.29, p=2.55x10-5) using

the Spearman ranked correlation analysis. The findings that high levels of EZH2

expression and low levels of CASZ1 expression occur in the tumors of poor prognosis

NB patients suggests that PRC2 components repress CASZ1 expression in NB cells.

CASZ1 transcription is repressed by EZH2

To evaluate whether CASZ1 is a target of EZH2, lentiviral shRNAs were used to

knock down EZH2 expression in SMS-KCNR cells. The expression of EZH2 decreased

3-fold in EZH2-shRNA transfected SMS-KCNR cells, while the mRNA expression of

CASZ1 increased 2-fold (Fig 2A). A pharmacologic inhibitor of EZH2, 3-

Deazaneplanosin A (DZNep), which depletes EZH2 protein expression and is reported to

relieve EZH2-mediated gene suppression (34), was used to further to determine whether

Tumor suppressor genes are regulated by EZH2 in NB 10

CASZ1 is a target of EZH2. qRT-PCR showed that the mRNA expression of CASZ1

increased 2-fold after the DZNep treatment consistent with decreased EZH2 protein

expression and a decrease in the global level of H3K27me3 (Fig 2B). We also examined

CASZ1 expression in immortalized EZH2-/- MEF cells (30). Expression of CASZ1 was

increased 3-fold in EZH2-/- cells compared to EZH2+/+ MEF cells (Fig 2C). These data

indicate CASZ1 may be a target of EZH2.

CASZ1 is a direct target of PRC2 complex

To determine whether CASZ1 is directly regulated by EZH2, we first scanned the

CASZ1 gene using the UCSC genome browser, which showed that the CASZ1

transcriptional start site (TSS) region is enriched with H3K4me3 and H3K27me3 in

embryonic stem cells (Fig 3A). The enrichment of the bivalent mark of H3K4me3 and

H3K27me3 is commonly found on silenced genes critical for development (35). Based

on the location of H3K27me3 enrichment on the CASZ1 gene in ES cells, a series of

PCR primer sets were designed to probe the PRC2 complex binding sites on the region of

CASZ1 from 4,000 base pairs upstream to 20,000 base pairs downstream of its TSS (Fig

3A). Under steady-state conditions, ChIP assays indicated that the three subunits of PRC2

all bound and were enriched around the CASZ1 TSS in SMS-KCNR cells (Fig 3B).

Moreover in the same region there was enrichment of H3K27me3 and H3K4me3, which

is consistent with silencing or low expression of CASZ1 in the SMS-KCNR cells. The

ChIP data reveals CASZ1 is subject to silencing chromatin modifications and is an EZH2

target in NB cells.

Tumor suppressor genes are regulated by EZH2 in NB 11

HDAC inhibitor induces CASZ1 and represses PRC2

Since previous studies indicated that HDAC inhibitors could restore the

expression of EZH2-repressed genes (21), we treated four different NB cell lines

(Supplemental Table 1) with varying concentrations of depsipeptide, a Type 1 HDAC

inhibitor (36). The four different NB cell lines contain characteristic genetic alterations

found in NBs (Supplemental Table 1). After 24 hours treatment, CASZ1 mRNA

expression increased 2-20 fold in all cell lines evaluated (Fig 4A). The steady-state

expression levels of PRC2 components, EZH2 and SUZ12 decreased in depsipeptide

treated NB cells. While the global level of H3K27ac increased after depsipeptide

treatment, the global level of H3K27me3 was unchanged or even increased in SK-N-AS

and SH-SY5Y cells (Fig 4B). We evaluated the H3K27me3 demethylases, KDM6A and

KDM6B, in the depsipeptide treated cells and found that the steady-state protein

expression of KDM6A decreased after depsipeptide treated NB cells (Fig 4C). We failed

to detect significant levels of KDM6B in NB cells (data not shown). This suggests that a

compensating decrease in a H3K27 demethylase maintains the global levels of

H3K27me3.

We used ChIP assays to examine the binding of PRC2 complex, RNA polymerase

II and the status of H3 modifications on the CASZ1 gene after depsipeptide treatment.

Following depsipeptide treatment, the binding of PRC2 complex to the CASZ1 TSS

decreased, and the binding of RNA polymerase II increased (Fig 5A). This is consistent

with the increased CASZ1 expression after depsipeptide treatment (Fig 4A). Consistent

with the depsipeptide induced decrease in PRC2 binding there was also a decrease of

H3K27me3 and an enrichment of H3K4me3 on the CASZ1 TSS (Fig 5A). We further

Tumor suppressor genes are regulated by EZH2 in NB 12

probed the binding of PRC2 complex, RNA polymerase II and the status of H3K27me3,

H3K27ac and H3K4me3 in other NB cell lines (SH-SY5Y and NGP) following

depsipeptide treatment (Fig 5B). All core PRC2 components binding to CASZ1

decreased along with decreased enrichment of H3K27me3 after depsipeptide treatment.

Consistent with the decreased occupancy of PRC2 components and H3K27me3, there

was increased H3K4me3 and RNA polymerase II binding to the CASZ1 TSS. These data

demonstrate that EZH2 directly represses CASZ1 in NB cells.

EZH2 also represses other tumor suppressors in NB.

We further explored whether other known tumor suppressors in NB were also

repressed by EZH2. The expression of reported tumor suppressors, CLU (37), NGFR

(reviewed in (3)), RUNX3 (29), CHD5 (7, 8), RASSF1A (38) and TP73 (reviewed in

(10)) were examined in the shRNA-transfected SMS-KCNR cells. Decreasing EZH2

expression increased CLU, NGFR and RUNX3 expression (Fig 6A), while the levels of

CHD5, RASSF1A and TP73 expression did not change (data not shown). The expression

of CLU, RUNX3 and NGFR were all increased after depsipeptide treatment (Fig 6B).

ChIP assays demonstrate the EZH2 binding and H3K27me3 enrichment on the promoter

regions of these three genes were decreased, consistent with the respective increase in

their expression (Fig 6C). Our data indicate that EZH2 mediated repression contributes to

the reduced expression of several genes in NB that have tumor suppressor activity or

whose elevated expression is associated with a good prognosis.

Tumor suppressor genes are regulated by EZH2 in NB 13

Silencing of EZH2 decreases NB cell growth and induces neurite extensions.

To investigate the biological function of EZH2 in NB, lentiviral shRNA were

used to decrease EZH2 expression in SMS-KCNR cells. The growth of cells infected

with the EZH2-shRNA was decreased to 20% after 3 days compared with control shRNA

infected cells (Fig 7A). Congruously NB cells displayed a concentration-dependent

decrease in cell survival after 4-day DZNep treatment (Fig 7B). There was also an

increase in cells with neurite-like processes in the EZH2 shRNA infected cells (Fig 7A)

and similar morphologic changes were observed in DZNep treated cells (Fig 7B).

Cell cycle analysis of DZNep treated KCNR cells indicated a 4-fold increase in

the subG1 phase of the cell cycle and a decrease in the growth fraction (S/G2-M) (Fig

7C). To assess whether DZNep-induced cell death via a caspase-dependent apoptotic

pathway, we evaluated caspase-3/7 activities. There was up to a 2-fold increase in

caspase 3/7 activities in DZNep-treated NB cells (Fig 7D). Incubation of cells with a pan

caspase inhibitor (Z-VAD-FMK) partially blocked the DZNep mediated decrease in NB

cell survival (Fig 7E). These data indicate that the DZNep induced increases in cell death

are partially due to induction of caspase-dependent apoptotic pathways. A preliminary

study to explore whether DZNep inhibited NB xenograft tumor growth indicates a

statistically significant (P<0.05) reduction in the growth of NB tumors in mice treated

with DZNep (Fig 7F).

Discussion

In this study, we find relatively high expression of EZH2 in more undifferentiated

or stroma poor NB tumors and high EZH2 expression is detected in the tumors of patients

Tumor suppressor genes are regulated by EZH2 in NB 14

with worse prognoses. We also find that the NB tumor suppressor genes, CASZ1, CLU,

NGFR and RUNX3 are direct targets of H3K27me3 mediated gene silencing by EZH2.

HDAC inhibition increases CASZ1 expression and decreases binding of PRC2 and the

H3K27me3 enrichment on the CASZ1 promoter. Our findings suggest EZH2-mediated

H3K27me3 is an important repressive mark of tumor suppressors in NB, and it also

indicates that EZH2 cooperates with histone deacetylases for effective repression of its

targets.

Over-expression of EZH2 is found in a number of cancers including those of

prostate (21), breast (28), as well as Ewings sarcoma (24), glioblastoma (25) and

melanoma (39, 40). Previous studies indicate that EZH2 is a prognostic biomarker in

breast (23) and prostate cancer (22, 41). We do find that there is an association between

EZH2 mRNA levels and NB prognosis but it is not as robust as previously described

prognostic markers in NB. However, the finding of EZH2 association with tumor

histology provides a clue as to the potential mechanism responsible for the variable

differentiation status of NB tumors. This is consistent with the report that EZH2 blockade

induces neural differentiation-associated genes in Ewings sarcoma (24), a tumor of

undifferentiated mesenchymal origin.

In this study, we used the newly identified NB tumor suppressor CASZ1 to probe

how EZH2 affects target genes and to shed light on mechanisms of histone methylations

that regulate tumor suppressor genes expression in NB. Recently we have published the

evidence supporting a tumor suppressor function of CASZ1 in NB (11). CASZ1 is a gene

localized in the 1p36, which is a region frequently deleted in the various kinds of cancers.

Over-expression of CASZ1 in NB cells decreases cell growth, cell migration, anchorage

Tumor suppressor genes are regulated by EZH2 in NB 15

independent growth and NB tumor growth in xenograft models (11). In clinical NB tumor

samples, low CASZ1 expression correlates with poor prognosis and high CASZ1

expression is found in the NB tumors with a differentiated histopathology. Since tumor

suppressors also require a functional loss of expression, our finding that EZH2 represses

CASZ1 expression in NB cell lines with 1pLOH (SMS-KCNR) as well as intact 1p (SH-

SY5Y) provides a mechanism to account for the functional loss of CASZ1 expression.

Using several model systems we provide evidence that CASZ1 is a direct target of EZH2.

Moreover, we find that the tumor suppressors, CLU, RUNX3 and NGFR, are also

repressed by EZH2 in NB. While EZH2 may also increase expression of genes associated

with NB tumorigenesis, we did not find significant increases in MYCN, Cyclin D1

(CCND1), PHOX2B, or NTRK2 mRNA levels when EZH2 is over-expressed (C. Wang

unpublished data). This suggests that EZH2 activity may represent a broad mechanism of

suppression of genes already noted to be important in NB tumor biology.

There are numerous different mechanisms reported to regulate the EZH2

expression and function, which could lead to the over-expression or increased activity of

EZH2 in NB. The genomic loss of microRNA-101, which inhibits EZH2 expression,

leads to prostate tumor (42) and this genomic region may be depleted via 1pLOH in NB.

Also, EZH2 expression is induced by E2F1 and repressed by activated Rb (43), and its

activity is stimulated by CDK1/2 (44). Over-expression of cell cycle genes is found in

NB tumors with the unfavorable prognoses (33) and over 75% of NB tumors have

elevated levels of cyclin D (45). In lymphoma, MYC is reported to stimulate EZH2

expression by repression of miR-26a (46). Accordingly the deregulation of MYCN,

which occurs in 20% NB patients (2) may be involved in the deregulation of EZH2. The

Tumor suppressor genes are regulated by EZH2 in NB 16

EZH2 locus on 7q35-36 is amplified in 10-15% tumors (43) and gain of the entire

chromosome 7 or 7q is detected in over 50% of NB tumors (47). Therefore, increased

DNA copy number could also contribute to increased EZH2 expression in a subset of NB

tumors. Preliminary evaluation of available microarray databases does not reveal a

significant association of EZH2 mRNA with MYCN. However, qRT-PCR analyses are in

progress to ascertain whether there is an association with other known prognostic markers

in NB. The complexity of potential alterations leading to dysregulation of EZH2

warrants a more detailed analysis of EZH2 regulatory mechanisms in NB tumors.

Our future experiments are aimed at determining all EZH2 targets in NB cells

using unbiased genomic strategies that include microarray analyses of NB cells with

silencing of EZH2 using shRNA and ChIP-seq to map targets. While HDAC inhibitors

are traditionally understood to reduce deacetylation, allowing unrestrained acetylation to

open chromatin and thereby increase gene expression, studies that have looked at array

data or at gene families have shown that approximately the same number of genes are

repressed as are induced (48). It should be possible with these future studies to identify

whether H3K27me3 (via EZH2 or another methytransferase) is involved in the repression

of genes after depsipeptide treatment.

Recently, studies focusing on DZNep as an epigenetic cancer therapy drug have

been reported in breast cancer (34), glioblastoma (49) and acute myeloid leukemia (50).

Our studies show that inhibition of EZH2 by gene silencing or pharmacologic inhibition

using DZNep leads to inhibition of NB cell growth and morphologic differentiation of

NB cells. This suggests that inhibition of EZH2 function or expression may be an

important drug targeting strategy for NB.

Tumor suppressor genes are regulated by EZH2 in NB 17

Acknowledgments

We thank Rogier Versteeg and Richard Volckmann (Academic Medical Center,

Department of Human Genetics, Amsterdam, the Netherlands) for making the R2

bioinformatics database publically available. We thank Doo-Yi Oh, Stanley He and other

lab members for their thoughtful review of the study and Lauren Marks for her support of

the Cell & Molecular Biology Section. This research was supported and funded by the

Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer

Research.

Tumor suppressor genes are regulated by EZH2 in NB 18

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Figure Legends

Fig 1. The level of EZH2 expression predicts the clinical outcome of NB patients.

A. EZH2 is highly expressed in more aggressive NB tumors comparing with the

differentiated neuroblastic tumors, ganglioneuroblastoma and ganglioneuroma (Left

panel, p=4.12x10-8), and high EZH2 expression is associated with stoma poor tumors

(Right panel). The graph is downloaded from Oncomine microarray database.

B. Kaplan-Meier survival plots were downloaded from R2 microarray analysis and

visualization platform. Patients with higher EZH2 and CASZ1 expression are highlighted

in blue, while patients with lower EZH2 or CASZ1 expression are highlighted in red.᧤

EZH2, p= 0.00042; CASZ1, p=0.0011).

C. EZH2 and CASZ1 expression is reverse correlated in NB clinical tumors and NB cell

lines. The microarray expression data were downloaded from Oncogenomics Section

Data center (http://pob.abcc.ncifcrf.gov/cgi-bin/JK). Log2 mean value is as the cut-off

“0”, lower expression comparing with the mean value is negative number and colored

blue, while higher is positive and colored red. The color codon is showed in color bar.

Fig 2. Genetic and pharmacologic inhibition of EZH2 expression leads to increase in

CASZ1 mRNA expression.

A. qRT-PCR measures the relative mRNA expression of EZH2 and CASZ1 in EZH2-

shRNA-transfected KCNR (shRNA) comparing with non-target shRNA-transfected cells

(Ctrl). The EZH2 protein expression and H3K27me3 status were detected by western

blot. H3 served as loading control.

Tumor suppressor genes are regulated by EZH2 in NB 26

B. SMS-KCNR cells were treated with 0.5uM DZNep for 48 hours. The mRNA

expression of CASZ1 was measured using qRT-PCR, and western blot examined protein

expression as indicated.

C Relative mRNA expression of EZH2 and CASZ1 were measured by qRT-PCR in

EZH2 knock out (EZH2-/-) and control MEF (EZH2+/+) cell lines, and western blot

showed the proteins expression as indicated.

Fig 3. PRC2 complex binds to the CASZ1 promoter

A. Schematic representation of CASZ1 locus, transcripts, coded proteins, CpG islands

and histone modifications, which was downloaded and modified from UCSC Genome

Browser. The box frames the genomic section of CASZ1 expanded below. Numbered

arrows indicate the genomic regions analyzed for PRC2 binding and histone

modifications using ChIP assays.

B. ChIP-PCR assays revealed the binding of PRC2 components and the indicated histone

marks, at CASZ1 locus in SMS-KCNR cells under steady-state conditions. The

enrichment of examined histone modifications and the PRC2 components in the indicated

regions were examined by qRT-PCR and plotted relative to input DNA.

Fig 4. HDACi depsipeptide induces CASZ1 and represses PRC2 complex.

A. NB cells were treated with different concentrations of depsipeptide for 24 hours. The

relative CASZ1 expression is determined by qRT-PCR.

B. Western blot showed the protein expression of EZH2, SUZ12 and the status of global

H3K27me3 and H3K27ac after treatment with depsipeptide (24hrs).

Tumor suppressor genes are regulated by EZH2 in NB 27

C. Western blot showed the protein expression of KDM6A after treatment with

depsipeptide (24hrs). Desitometric analysis was performed using Image J. The relative

expression of KDM6A (normalized to Ponceau S staining of Histone 3) in depsipeptide

treated cells is normalized to the untreated control, normalized KDM6A values and

expressed as relative density units (RDU).

Fig 5. ChIP analysis indicates that the binding of PRC2 complex is attenuated by

HDACi depsipeptide treatment.

A. SMS-KCNR cells were treated with 2ng/ml depsipeptide for 24 hours. ChIP assays

reveal the relative enrichment of indicated histone marks, RNA polymerase II (pol II) and

the binding of PRC2 components at the CASZ1 locus in depsipeptide treated (KCNR

Depsi, grey line) or steady-state or control KCNR cells (KCNR Ctrl, black line).

B. SH-SY5Y and NGP cells were treated with 2ng/ml depsipeptide for 24 hours (control,

black; depsipeptide treated, grey). ChIP assays indicated the H3K27me3 status, PRC2

complex binding (primer No. 4) and H3K4me3, H3K27ac status and RNA polymerase II

(pol II) binding (primer No. 1) on the CASZ1 promoter region.

Fig 6. EZH2 also directly represses expression of NB tumor suppressor genes.

A. qRT-PCR measured the mRNA relative expression of EZH2, CLU, NGFR and

RUNX3 in EZH2-shRNA-transfected KCNR (EZH2-shRNA) compared to non-target

shRNA-transfected cells (Ctrl).

B. The relative CLU, NGFR and RUNX3 expression levels after treatment of NB cells

with depsipeptide or control solvent were determined by qRT-PCR.

Tumor suppressor genes are regulated by EZH2 in NB 28

C. ChIP assays revealed the indicated histone marks, the binding of RNA polymerase II

(pol II) and EZH2 at CLU, NGFR and RUNX3 promoters in depsipeptide treated (Depsi,

grey) or non-treated KCNR cells (Ctrl, black).

Fig 7. Decrease of EZH2 affects the cell growth and induces the neurite growth.

A. Cell survival in KCNR cells after 3-day infection with EZH2 or non-target shRNA

lentivirus was assessed using a Cell-Titer Blue assay (left panel). The percentage of

surviving cell was normalized by the absorbance value of the non-target shRNA infected

cells (Control). Representative images (×200) of the non-target shRNA infected cells

(Ctrl, middle panel), EZH2 shRNA infected cells (EZH2 shRNA, right panel).

B. KCNR cells were treated with different concentration of DZNep for 96 hours. MTS

assay was used to detected cell survival (left panel). The percentage of surviving cells

was normalized by the absorbance value of the non-treated cells. Representative images

(×200) of the non-treated cells (Ctrl, middle panel) and 0.5uM DZNep treated cells

(DZNep 0.5uM, right panel).

C. KCNR cells were treated with different concentration of DZNep for 96 hours. The

cells were stained with propidium iodide and analyzed by flow cytometry. The data

showed percentage of events in sub-G1, G1 and S/G2-M phase.

D. Caspase 3/7 activities were assessed after 48 hours with different concentration of

DZNep in KCNR cells. The percentage of caspase 3/7 activities was graphed after

normalization to non-treated cells.

Tumor suppressor genes are regulated by EZH2 in NB 29

E. KCNR cells were treated with 5uM DZNep in the absence or presence of 100uM pan

caspase inhibitor, Z-VAD-FMK, for 72 hours. The percentage of surviving cells was

graphed after normalization to untreated control.

F. Mice were treated with or without DZNep (2.5mg/kg) twice a day, 3 days per week for

4 weeks. The mean tumor volumes are plotted using the SEM (standard error of the

mean). The time points with significant differences (p<0.05) were indicated with asterisk.

Tumor suppressor genes are regulated by EZH2 in NB 30

EZH2 mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3 and NGFR

Chunxi Wang, Zhihui Liu, Chan-Wook Woo, et al.

Cancer Res Published OnlineFirst November 8, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-11-0961

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